Spatial mapping of molecular profiles of biological tissue samples

Abstract

A method is presented that enables the spatial mapping of nucleic acids of tissue samples with high resolution and without sacrificing the degree of multiplexing that is available from next-generation sequencing. The method is based on the application of patterns of barcoded oligonucleotides probes onto predefined locations in a region of interest in a tissue sample. Every nucleic acid analyzed can be allocated to a certain position inside the sample based on the barcode. Various printing technologies can be used and different ways of patterning can be employed, like a regular array with a certain pitch or alternatively an object-based patterning with defined regions of interest without shape constraints.

The invention claimed is: 
     
       1. A method for spatial detecting nucleic acids in a tissue sample comprising the steps of:
 separating the sample into at least a first layer and a second layer; 
 applying at least one imaging label to the first layer; 
 imaging the first layer; 
 identifying at least one region of interest (ROI) within the sample from the image of the first layer and applying the ROI to the second layer; 
 applying at least one species of oligonucleotide probes onto predefined locations within the ROI, said predefined locations defined by a full separation mask, wherein the full separation mask includes a lattice separation mask, and allowing the oligonucleotide probes to bind to the nucleic acids of the sample, wherein the oligonucleotide probes comprise a barcode sequence; 
 extracting the nucleic acid-oligonucleotide probes complexes; 
 sequencing the extracted nucleic acid molecules; 
 correlating the sequenced nucleic acid molecules to the initial location of the corresponding targeted nucleic acid molecules within the ROI to generate a spatial distribution of the targeted nucleic acid molecules, wherein each location is identified by one or more species of oligonucleotide probes bound in the step of applying at least one species of oligonucleotide probes. 
 
     
     
       2. The method according to  claim 1 , wherein prior to the step of sequencing the extracted nucleic acid molecules, DNA molecules are generated from the nucleic acid-oligonucleotide probes complexes via DNA amplification. 
     
     
       3. The method according to  claim 2 , wherein the generation of DNA molecules occurs after a reverse transcription reaction. 
     
     
       4. The method according to  claim 2 , wherein in the step of applying at least one species of oligonucleotide probes, the binding of the oligonucleotide probes to the nucleic acids of the sample occurs via hybridization and wherein the oligonucleotide probes are used as primers. 
     
     
       5. The method according to  claim 1 , wherein in the step of applying at least one species of oligonucleotide probes, the binding of the oligonucleotide probes to the nucleic acids of the sample occurs via ligation. 
     
     
       6. The method according to  claim 5 , wherein the step of correlating the sequenced nucleic acid molecules further comprises correlating the spatial distribution of the targeted nucleic acid molecules with an image of the ROI or with an image of the tissue sample in which the ROI was identified obtained before or after the step of applying at least one species of oligonucleotide probes. 
     
     
       7. The method according to  claim 6 , wherein the method further comprises providing a two-dimensional spatial map to visualize the spatial distribution of the targeted nucleic acid molecules. 
     
     
       8. The method according to  claim 7 , wherein the method further comprises overlaying the two-dimensional spatial map with the image of the ROI or with the image of the tissue sample in which the ROI was identified obtained before or after the step of applying at least one species of oligonucleotide probes. 
     
     
       9. The method according to  claim 1 , wherein at least two different species of oligonucleotide probes are bound to one targeted nucleic acid molecule in the step of applying at least one species of oligonucleotide probes, and wherein the unique combination of the at least two different species of oligonucleotide probes is used to identify the location of the targeted nucleic acid molecules within the ROI. 
     
     
       10. The method according to  claim 1 , wherein at least one of the species of oligonucleotide probes that bind to one nucleic acid molecule comprises a generic sequence, wherein optionally the generic sequence is complementary to the targeted nucleic acid. 
     
     
       11. The method according to  claim 10 , wherein at least one of the species of oligonucleotide probes that bind to one nucleic acid molecule comprises an additional sequence, wherein the additional sequence is a purification sequence or a primer alignment sequence. 
     
     
       12. The method according to  claim 1 , wherein in the step of applying at least one species of oligonucleotide probes, the oligonucleotide probes are applied onto the predefined locations by liquid transfer technologies, preferably by contact printing techniques or non-contact printing techniques, such that the applied oligonucleotide probes do not intermix between different predefined locations. 
     
     
       13. The method according to  claim 1 , wherein the sample is a histopathological specimen, preferably a deparaffinised formalin-fixed paraffin-embedded (FFPE) sample, a fresh frozen (FF) sample or a fresh sample, or a cytology sample. 
     
     
       14. The method of  claim 1 , wherein the at least one imaging label applied to the first layer does not interfere with any of the steps of applying at least one species of oligonucleotide probes, extracting the nucleic acid-oligonucleotide probes complexes, or sequencing the extracted nucleic acid molecules performed on the second layer. 
     
     
       15. A method for spatial detecting nucleic acids in a tissue sample comprising the steps of:
 separating the sample into at least a first layer and a second layer; 
 applying at least one imaging label to the first layer; 
 imaging the first layer; 
 identifying at least one region of interest (ROI) within the sample from the image of the first layer and applying the ROI to the second layer; 
 applying at least one species of oligonucleotide probes onto predefined locations within the ROI, said predefined locations defined by a freeform separation mask having an irregular shape based on the shape of the ROI, and allowing the oligonucleotide probes to bind to the nucleic acids of the sample, wherein the oligonucleotide probes comprise a barcode sequence; 
 extracting the nucleic acid-oligonucleotide probes complexes; 
 sequencing the extracted nucleic acid molecules; 
 correlating the sequenced nucleic acid molecules to the initial location of the corresponding targeted nucleic acid molecules within the ROI to generate a spatial distribution of the targeted nucleic acid molecules, wherein each location is identified by one or more species of oligonucleotide probes bound in the step of applying at least one species of oligonucleotide probes. 
 
     
     
       16. The method according to  claim 15 , wherein prior to the step of sequencing the extracted nucleic acid molecules, DNA molecules are generated from the nucleic acid-oligonucleotide probes complexes via DNA amplification. 
     
     
       17. The method according to  claim 16 , wherein the generation of DNA molecules occurs after a reverse transcription reaction. 
     
     
       18. The method according to  claim 16 , wherein in the step of applying at least one species of oligonucleotide probes, the binding of the oligonucleotide probes to the nucleic acids of the sample occurs via hybridization and wherein the oligonucleotide probes are used as primers. 
     
     
       19. The method according to  claim 15 , wherein in the step of applying at least one species of oligonucleotide probes, the binding of the oligonucleotide probes to the nucleic acids of the sample occurs via ligation. 
     
     
       20. The method according to  claim 19 , wherein the step of correlating the sequenced nucleic acid molecules further comprises correlating the spatial distribution of the targeted nucleic acid molecules with an image of the ROI or with an image of the tissue sample in which the ROI was identified obtained before or after the step of applying at least one species of oligonucleotide probes. 
     
     
       21. The method according to  claim 20 , wherein the method further comprises providing a two-dimensional spatial map to visualize the spatial distribution of the targeted nucleic acid molecules. 
     
     
       22. The method according to  claim 21 , wherein the method further comprises overlaying the two-dimensional spatial map with the image of the ROI or with the image of the tissue sample in which the ROI was identified obtained before or after the step of applying at least one species of oligonucleotide probes. 
     
     
       23. The method according to  claim 15 , wherein at least two different species of oligonucleotide probes are bound to one targeted nucleic acid molecule in the step of applying at least one species of oligonucleotide probes, and wherein the unique combination of the at least two different species of oligonucleotide probes is used to identify the location of the targeted nucleic acid molecules within the ROI. 
     
     
       24. The method according to  claim 15 , wherein at least one of the species of oligonucleotide probes that bind to one nucleic acid molecule comprises a generic sequence, wherein optionally the generic sequence is complementary to the targeted nucleic acid. 
     
     
       25. The method according to  claim 24 , wherein at least one of the species of oligonucleotide probes that bind to one nucleic acid molecule comprises an additional sequence, wherein the additional sequence is a purification sequence or a primer alignment sequence. 
     
     
       26. The method according to  claim 15 , wherein in the step of applying at least one species of oligonucleotide probes, the oligonucleotide probes are applied onto the predefined locations by liquid transfer technologies, preferably by contact printing techniques or non-contact printing techniques, such that the applied oligonucleotide probes do not intermix between different predefined locations. 
     
     
       27. The method according to  claim 15 , wherein the sample is a histopathological specimen, preferably a deparaffinised formalin-fixed paraffin-embedded (FFPE) sample, a fresh frozen (FF) sample or a fresh sample, or a cytology sample. 
     
     
       28. The method of  claim 15 , wherein the at least one imaging label applied to the first layer does not interfere with any of the steps of applying at least one species of oligonucleotide probes, extracting the nucleic acid-oligonucleotide probes complexes, or sequencing the extracted nucleic acid molecules performed on the second layer.